Nonrandom Double Strand Cleavage of DNA by a Monofunctional Metal Complex: Mechanistic Studies

ions from deoxyribose produces thiobarbituric-reactive material. In addition, the quantitation of OH• radical by rhodamine must be considered a lower limit on the amount actually formed, since other OH• reaction pathways (e.g., reaction with buffer, self-quenching) have not been quantitated. Because active oxidants other than hydroxy radical (such as metal-oxo species) can lead to ribose degradation products indistinguishable from those formed by OH•, we examined our reaction in the presence of three known hydroxy radical quenchers, thiourea, DMSO, and ethanol (Figures 7 and 8). In all cases, the reaction is suppressed, regardless of whether both activating agents or ascorbate alone are used. This suggests that the active oxidant is indeed hydroxy radical. However, since the quenchers used are all also ligands, inhibition of reaction may occur not by radical quenching, but by blocking of the coordination site on copper that is involved in radical formation. As noted above, the visible spectra of 1 in the presence and absence of DMSO or ethanol are identical (Figure 1), so suppression of reaction by coordination appears not to occur with these quenchers, but by true OH• quenching. Thiourea, however, effects a change in the visible spectrum of 1, and may inhibit reaction in part by blocking the coordination site at which the OH• precursor binds. Hydroxy radicals are generated by a variety of chemical and physical processes. Transition metal mediated OH• production is known to occur by a number of routes; two well-known pathways are the Fenton mechanism and the Haber-Weiss mechanism. In the Fenton mechanism, the metal ion acts as a catalyst for hydroxy radical generation from hydrogen peroxide: The Haber-Weiss reaction (eq 4)21 involves the reaction of superoxide with H2O2 to produce dioxygen, hydroxide, and OH•: The production of O2 during reaction does not preclude a Fenton mechanism, however, since dismutation of O2 to dioxygen and H2O2 can occur: We attempted to quantitate the amount of superoxide formed during the reaction using a standard reductive colorimetric assay for O2 (nitro blue tetrazolium22,23 ). However, in initial studies, we found that, under anaerobic conditions, both sodium ascorbate or reduced 1 have the ability to reduce the dye molecule directly. Similar problems with competing redox reactions have been noted with colorimetric assays for O2. We then attempted to detect the formation of superoxide during reaction by adding SOD to the reaction mixture. Only reactions which proceed through an O2 intermediate should be affected by the presence of the enzyme. As can be seen in Table 2, reaction is effectively quenched by the presence of SOD, indicating that O2 is an intermediate in the production of OH•. The role of dioxygen in radical trapping by deoxyribose was then investigated by conducting the reaction under anaerobic conditions with either ascorbate activation or tandem ascorbate/ H2O2 activation. As can be seen from the data in Table 2, the exclusion of molecular oxygen under either set of activation conditions effectively suppresses the formation of trapped product. While these results are consistent with the requirement of O2 and O2 as participants in OH• formation, they do not constitute confirmation. The suppression of reaction under anaerobic conditions may result not from the failure to abstract a proton from deoxyribose but rather from the lack of O2 to react with the deoxyribose radical formed Via proton abstraction, which leads to thiobarbituric-reactive products.25 Degradation of rhodamine B by OH•, however, does not require O2 in the degradation pathway. Anaerobic reaction of OH• formation by 1 does not produce any degradation of rhodamine (Figure 8). This confirms the requirement of O2 as reactant in the generation of hydroxy radical by 1 in the presence or absence of H2O2.